Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157.

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.

Attachment of different Escherichia coli strains to cultured rumen epithelial cells.

The attachment to fully characterized primary rumen epithelial cell cultures of Escherichia coli strains isolated from different animal species and expressing F1-F4 or F17 fimbriae was examined. As the cell cultures contained stratified (keratinized) and non-stratified (non-keratinized) cells which grew either confluently or non-confluently, the strength of attachment of the different bacterial strains was assessed in relation to the differentiation state of the cells. Thus, strains having F1 fimbriae attached to all types of cultured cells, while strains with F2 and F3 fimbriae did not bind at all. E. coli strains having F4 or F17 fimbriae attached only to non-keratinized cells, particularly to confluent areas. As membrane glycosylation is known to change with differentiation (keratinization), our results suggest that the attachment of fimbriated E. coli strains which were capable of binding to rumen cells was more likely to be dependent on differentiation than the host specificity of the bacteria.

Characterization of Na+/H+ exchange in sheep rumen epithelial cells kept in primary culture.

The study aimed at testing whether Na+/H+ exchange activity is maintained during isolation and cultivation of rumen epithelial cells (REC) and whether Na+/H+ exchange is altered during proliferation and differentiation. REC were isolated from sheep rumen epithelium by fractional trypsinization and further cultivated for up to 21 days. Characteristics of Na+ uptake into cultured REC were the same as previously described for the Na+/H+ exchange in the intact rumen epithelium. Na+ uptake was increased by intracellular acidification and was inhibited by high doses of amiloride. Amiloride inhibited 3H-thymidine incorporation into REC at concentrations similar to those found for the inhibition of Na+ uptake. Fetal calf serum, a growth factor, stimulated amiloride-sensitive Na+ uptake. We therefore conclude that cultured rumen epithelial cells express Na+/H+ exchange and that the activity of the exchange is at least correlated to the intensity of proliferation.

Lectins as markers of rumen epithelial cell differentiation.

Lectins of different carbohydrate specificities (GNA (Galanthus nivalis), con A (Canavalia ensiformis), VFL (Vicia faba), PSL (Pisum sativum), LCA (Lens culinaris), PNA (Arachis hypogaea; with or without prior neuraminidase treatment), WGA (Triticum vulgare), SBA (Glycine max), UEA-I (Ulex europaeus), LPA (Limulus polyphemus), BS-I B4 (Bandeiraea simplicifolia, isolectin B4)) were explored for use as differentiation markers of rumen epithelial cells in vivo and in vitro. Lectins specific for mannose (GNA), mannose/glucose (con A, VFL, PSL and LCA), N-acetylglucosamine (WGA) or for N-acetylneuraminic acid (LPA) reacted generally with all types of rumen epithelial cell from both rumen tissue and cell culture. They were, therefore, not suitable markers of epithelial differentiation. SBA was unsuitable because, although it reacted with both tissue and cultured rumen epithelial cells, it was also bound to non-stratified areas of primary rumen epithelial cell cultures. Both BS-I B4 and PNA (after neuraminidase treatment) had to be ruled out because they did not react with differentiated rumen tissue epithelial cells, although they did bind to both stratified and non-stratified cultured cells. In contrast, UEA-I reacted strongly with differentiated rumen epithelial cells both from rumen tissue and cell cultures and therefore appears to be a good general marker for rumen epithelial cell differentiation.

Acute metabolic and hormonal effects of intravenously administered sodium n-butyrate in untreated and alloxan-diabetic sheep.

Experimental diabetes was induced in 4 wethers of the Mutton Merino breed by intravenous injection of alloxan (75 mg.kg-1) in order to determine its impact on plasma glucose, immunoreactive insulin, free fatty acids (FFA), cholesterol, phospholipids, triglycerides, D-(-)-3-hydroxybutyrate (D-3-HB), bilirubin and aspartate aminotransferase (ASAT) as well as on the changes of these parameters brought about by an intravenous infusion of sodium n-butyrate (1 mmol.kg-1). Alloxan administration caused a significant elevation of plasma glucose, FFA, triglycerides, cholesterol, phospholipids, D-3-HB and bilirubin and a decrease of the level of immunoreactive insulin. The increase in glucose level brought about by a bolus injection of sodium n-butyrate in untreated sheep did not appear in alloxanized animals. Thus, it is suggested that the lack of hyperglycaemic response in diabetic sheep was due to the absence of liver glycogen stores. Unexpectedly in alloxan-diabetic sheep, a decrease in the plasma level of FFA occurred after the administration of sodium n-butyrate. Therefore, it may be assumed that beside insulin other factors may contribute to the decrease of FFA under these conditions.

The inhibitory action of sodium butyrate on the growth of KB, MMT and RPMI cells.

Three tumor cell lines (KB, MMT and RPMI) established from epithelial tissues were treated for 24 h with sodium butyrate (BU), the BU concentrations giving rise to 50% inhibition of [3H]thymidine incorporation were 2.0, 0.3, 0.2 mmol/L, respectively, for the KB, MMT and RPMI cell lines. Studies with [14C]BU have shown that, at similar degrees of inhibition of [3H]thymidine incorporation, the intracellular concentrations of BU are very close for all three cell types, despite the dissimilarity of the extracellular BU concentrations. These results imply that the BU sensitivity of the cells does not depend on the inhibition of thymidine incorporation, but on their BU uptake. The [3H]thymidine incorporation of MMT cells exposed to 5 mmol/L BU for 72 h returned to normal within the next 48 h. The same treatment accounted for about an 80-90% decrease in the cloning efficiency and tumourigenicity of MMT cells. These findings indicate that BU pretreatment inhibits DNA synthesis temporarily, while other parameters related to the cell growth, such as cloning efficiency and tumourigenicity, are durably influenced by BU pretreatment.

Effects of butyrate and insulin and their interaction on the DNA synthesis of rumen epithelial cells in culture.

Rumen epithelial cells (REC) were incubated in the presence of various concentrations of butyrate or insulin or with both of them, to obtain information on their effect on the DNA synthesis of cultured cells. The 24-h values of 3H-thymidine incorporation into cellular DNA were measured in the presence of butyrate, insulin or butyrate plus insulin. While butyrate reduced DNA synthesis, insulin produced an increase over the control. Combined butyrate plus insulin treatment influenced the incorporation of label in accordance with the relative proportion of these two substances.

The effects of butyrate and glucagon on the proliferation of ruminal epithelial cells in culture.

When the rate of ruminal epithelial cell proliferation was measured on the basis of 3H-thymidine incorporation into the cellular DNA, butyrate dose-dependently reduced 3H-thymidine incorporation. In contrast, glucagon at 10 and 100 pg/ml had a slight stimulatory effect on the incorporation, but only in the absence of butyrate.

Influence of sodium butyrate on HeLa cell morphology and proliferation.

The effect of one-week exposure to sodium butyrate on HeLa S3 cell cultures was studied with special regard to influence on prekeratin synthesis, by comparison to cultures similarly treated with the known proliferation inhibitor hydroxyurea, and not treated. Like hydroxyurea, sodium butyrate inhibited cell proliferation to a considerable degree, but accounted additionally for an increase in membrane-bound alkaline phosphatase activity, cellular prekeratin synthesis, tonofilament number, and filament bundle formation. These phenomena unequivocally indicate that sodium butyrate acted as a specific stimulator of Hela (epithelial) cell differentiation. Similar differentiation phenomena can be observed during early spontaneous keratinization of the stratified horny epithelium.

Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme.

Rabbits immunized with low-activity ruminal carbonic anhydrase (RCA) isoenzyme, extracted from ruminal epithelial cells isolated by digestion with trypsin, yielded anti-RCA sera which reacted specifically with bovine RCA in double agar gel diffusion and immunoelectrophoretic tests, but failed to cross-react with bovine erythrocyte CA. The localization of RCA was identified in histological sections and isolated ruminal epithelial cell preparations by indirect immunofluorescence and immunoperoxidase tests as the basal, spinosum and granulosum layers of ruminal mucous epithelium.

Culture of epithelial cells from bovine ruminal mucosa.

A method is reported for the primary in vitro culture of epithelial cells derived from bovine ruminal mucosa. That the cultures of ruminal epithelial cells consisted exclusively of stratum spinosum, stratum basale and stratum granulosum was confirmed by immunoperoxidase and immunofluorescence staining using carbonic anhydrase isoenzyme as a marker.